Combined PD-L1 and TIM-3 blockade improves the expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy

Abstract

Background. The stimulation and expansion of antigen-specific T cells ex vivo enables the targeting of a multitude of cancer antigens. However, clinical scale T-cell expansion from rare precursors requires repeated stimulations ex vivo leading to T-cell terminal effector differentiation and exhaustion that adversely impact therapeutic potential. We leveraged immune checkpoint blockade relevant to antigen-specific CD8+ human T cells to improve the expansion and function of T cells targeting clinically relevant antigens. Methods. A clinically-compliant protocol relying on peptide-pulsed monocyte-derived dendritic cells and cytokines was used to expand antigen-specific CD8+ targeting the oncogenic Epstein-Barr virus (EBV) and the tumor associated antigen (TAA) Wilms Tumor 1 (WT1) protein. The effects of antibody-mediated blockade of immune checkpoints applied to the cultures (T-cell expansion, phenotypes and function) were assessed at various time points. Genomic studies including single cell RNA (scRNA) sequencing and T-cell receptor sequencing was performed on EBV-specific T cells to inform about the impact of immune checkpoint blockade on the clonal distribution and gene expression of the expanded T cells. Results. Several immune checkpoints were expressed early by ex vivo expanded antigen-specific CD8+ T cells, including PD-1 and TIM-3 with co-expression matching evidence of T-cell dysfunction as the cultures progressed. The introduction of anti-PD-L1 (expressed by the dendritic cells) and anti-TIM-3 antibodies in combination (but not individually) to the culture led to markedly improved antigen-specific T-cell expansion based on cell counts, fluorescent multimer staining and functional tests. This was not associated with evidence of T-cell dysfunction when compared to T cells expanded without immune checkpoint blockade. Genomic studies largely confirmed these results, showing that double blockade does not impart particular transcriptional programs or patterns on TCR repertoires. However, our data indicate that combined blockade may nonetheless alter gene expression in a minority of clonotypes and have donor-specific impacts. Conclusions. The manufacturing of antigen-specific CD8+ T cells can be improved in terms of yield and functionality using blockade of TIM-3 and the PD-L1/PD-1 axis in combination. Overcoming the deleterious effects of multiple antigenic stimulations through PD-L1/TIM-3 blockade is a readily applicable approach for several adoptive-immunotherapy strategies.

Neoantigen-specific stimulation of tumor-infiltrating lymphocytes enables effective TCR isolation and expansion while preserving stem-like memory phenotypes

“…6 20 21 54 55 Further ex vivo expansion of TILs by IL-2 or stimulation like the rapid expansion with OKT3 can exacerbate their already exhausted phenotypes. [32][33][34][35] Neoantigenic stimulation appears to address this issue by selectively expanding neoantigenreactive TILs, including rare neoantigen-reactive TIL clonotypes. When corresponding neoantigen-reactive TCRs were isolated from these rare neoantigen-reactive TIL clonotypes and tested (figure 2H and online supplemental figure S2-4), they demonstrated high avidity and specificity for neoantigens and antitumor efficacy in various in vitro (online supplemental figure S3B) or in vivo (figure 2H and online supplemental figure S2D) models, despite the notion that TCRs isolated following on June 9, 2024 by guest.…”

“…The conventional method for T-cell ex vivo expansion using OKT3 (an anti-CD3 antibody) and high dose interleukin 2 (IL-2), also known as the rapid expansion, 31 may cause outgrowth of bystander cells due to its non-specific nature. [32][33][34][35] In the current study, we tested whether neoantigen-reactive TILs could be stimulated in a neoantigen-specific fashion to achieve two main goals: first, sensitive identification of neoantigenreactive TCRs and second, development of TIL ACT products with high neoantigen reactivity. Using p53 and RAS family (ie, KRAS, NRAS and HRAS) mutations as a model, we demonstrate that neoantigenic stimulation could overcome the aforementioned challenges of the conventional TIL expansion by improving reactivities, phenotypes and functions of TILs.…”